C H A P T E R 3 Tendon Tissue Engineering K. Hampson*, N.R. Forsyth, A. El Haj and N. Maffulli Summary he successful healing of tendon injuries depends on numerous factors, including anatomical location, vascularity, skeletal maturity and the amount of tissue loss. Although spontaneous healing can T occur, this often results in the formation of scar tissue which is morphologically, biochemically and biomechanically different from healthy tendon tissue. This ultimately affects the functionality of the repaired tissue. Tendon tissue engineering aims to induce self-regeneration of the tendon tissue in vivo, or to produce a functional tissue replacement in vitro which can then be implanted into the body. The production of tendon tissue which is both viable and functional requires the generation of a uniaxially orientated matrix. The production and orientation of this matrix can potentially be altered by both biochemical and physical factors, and the combination of these two factors in a dose and time-dependent manner is potentially the key to successfully engineered tendons. This chapter reviews current strategies for tendon tissue engineering and the future challenges associated with this field. KEYWORDS: Scaffolds, Cell source, Mechanical stimuli, Growth factors, Gene therapy, Tissue engineering *Correspondence to: K. Hampson, ISTM, Keele University, Staffordshire, UK. E-mail: [email protected] Topics in Tissue Engineering, Vol. 4. Eds. N Ashammakhi, R Reis, & F Chiellini © 2008. Hampson et al. Tendon Tissue Engineering INTRODUCTION In the UK the National Health Service (NHS) treats thousands of damaged tendons each year, ranging from repetitive strain injuries (RSIs) to complete ruptures. Tendon injuries are difficult to manage and although spontaneous healing can occur this often results in the formation of scar tissue. Problems arise because the structure of scar tissue differs from healthy tissue which affects both the functionality of the repaired tissue, its movement and its strength [1]. Many tendon injuries occur in athletes and active people and the effect of having tendon tissue with reduced functionality can be devastating to their everyday lives. Current treatments, both conservative and surgical have shown limited success [2] , which demonstrates the need for tendon tissue engineering . STRUCTURE AND FUNCTION Tendon tissue is a type of connective tissue which physically binds muscles to skeletal structures [3] permitting locomotion and enhancing joint stability [4]. Tendon has a multi-unit hierarchical structure of collagen molecules, fibrils, fibre bundles, fascicles and tendon units [4] designed to resist tensile loads [5]. The organisation of the extracellular matrix (ECM) molecules of tendon at the micrometer and nanometer levels are the principal determinants of the physiological function and the mechanical strength of the tissue [4]. Microscopically, tendon has a crimped, waveform appearance which plays an important role in its mechanical properties [4]. The angle and length of the “crimp pattern” depends on the type of tendon, its anatomical site within the body and its location within the tendon tissue. The differences in the “crimp pattern” affect tendon's mechanical properties, and fibres which have a small crimp angle are mechanically weaker than those with a larger crimp angle [6]. MOLECULAR COMPOSITION Proteoglycans in the extracellular matrix enhance the mechanically properties of the tissue. Aggrecan helps to retain water within the ECM, increasing the tissues resistance to compression [7]. Decorin is thought to facilitate fibrillar slippage during mechanical deformation [8]. The concentration of proteoglycans varies within the tissue, and depends on the mechanical loading to which the tendon is exposed [9]. The proteoglycan content is higher in the areas which are subjected to compression, given the role of proteoglycans in resisting compression [7, 10]. Topics in Tissue Engineering, Vol. 4. Eds. N Ashammakhi, R Reis, & F Chiellini © 2008. 2 Hampson et al. Tendon Tissue Engineering Tendon tissue contains also glycoproteins, including fibronectin and tenascin-C. Fibronectin, an adhesive glycoprotein located on the surface of collagens [11], is involved the regeneration and repair of tendon [12]. It may also play a role in cell attachment to prevent cell removal due to gliding friction during tendon movement [13]. Tenascin-C is thought to be involved in the ECM network formation which contributes to the mechanical stability of the ECM in tendon tissue by interacting with both the collagen fibrils and the proteoglycan decorin [14]. Tenascin-C is not widely expressed in healthy musculoskeletal tissues, but almost exclusively at the sites subjected to heavy mechanical forces or requiring elastic properties, and is an elastic protein [15]. Tendon tissue also contains elastin, which comprises approximately 2% of the dry weight of tendon tissue [16]. The most abundant molecular component in tendon tissue is collagen type I. It constitutes approximately 60% of the dry mass of the tendon, and about 95% of the total tendon collagen content [17-19]. The remaining 5% of collagens consist mainly of collagens type III and V [4], with collagens type II, VI, IX, X and XI present in trace quantities [20]. Collagen type I molecules self assemble into highly organised fibrils which form collagen fibres [21, 22]. Collagens in the matrix are cross-linked, conferring them a high tensile strength and providing mechanical strength to the tendon tissue [23]. Collagen type III also forms fibrils, but these are smaller and less organised [24]. Although the overall cell content in tendon tissue is low [25], two main types of cells coexist, the tenocytes and the tenoblasts. Both types of these cells have mesenchymal origin. Tenoblasts are immature tendon cells, they are spindle shaped, and have numerous cytoplasmic organelles which reflect their high metabolic activity. These are the predominant cell type in tendon, and often appear in clusters within a localised pericellular region, devoid of collagen fibre anchorage. Tenoblasts mature into tenocytes, which have a fibroblastic morphology and have a much lower nucleus-to-cytoplasm ratio and a lower metabolic activity [26]. Tenocytes are terminally differentiated with a very limited proliferative capacity and are distributed throughout the tissue attached to collagen fibres [27]. Different populations of tenocytes have been identified in tendon tissue, based on their morphologies [28, 29]. However, these cell types have not yet been characterized and their exact function remains unclear [28]. Other types of cells present in tendon tissue are progenitor cells [30], endothelial cells, synovial cells and chondrocytes, although these are much less predominant [31]. There is also a subpopulation of Topics in Tissue Engineering, Vol. 4. Eds. N Ashammakhi, R Reis, & F Chiellini © 2008. 3 Hampson et al. Tendon Tissue Engineering myofibroblast-like, contractile cells present in normal tendon tissue. They are thought to be involved in the modulation of the contraction-relaxation of the muscle-tendon complex [32]. REQUIREMENT FOR TENDON TISSUE ENGINEERING There are approximately 2 x 10 5 tendon and ligament repairs performed annually in the U.S. [33]. These can occur through injury and trauma, commonly in the workplace and in sport, but also through overuse and ageing. The most commonly injured tendons are the Achilles and the patellar tendons, with pathology ranging from calcifying tendinopathy, partial tears, to complete ruptures [34, 35]. Injuries which present with pain, swelling, bruising and tearing of the tissue usually occur when the tendon has been under tensile load. Tendons typically affected in this way are the patellar tendon, long head of the biceps, and Achilles tendon. Tendon ruptures can also occur with no episode of a serious injury and little swelling. In these cases, pain and/or inability to play sports will be the major presenting complaint. These injuries often occur in the rotator cuff, extensor carpi radialis brevis or posterior tibial tendon [36]. Tendon injuries are difficult to manage, due to impaired healing, and frequently result in long-term pain and discomfort, which places a chronic burden on health care systems [1]. Successful healing of tendon injuries depends on numerous factors, including anatomical location, vascularity, skeletal maturity, and the amount of tissue loss. Although in most tissues the repair process involves the infiltration of blood cells, mature tendons are poorly vascularised [37-39], and tendon nutrition relies on synovial fluid diffusion rather than vascular perfusion [40]. Although spontaneous healing can occur, this often results in the formation of scar tissue which is morphologically, biochemically and biomechanically different from healthy tendon tissue. This ultimately affects the functionality of the repaired tissue [25]. Severe tendon injuries are difficult to manage because repair rarely results in tissue with fully restored function. Another problem which can occur is the imperfect integrative healing at tendon-tendon or tendon-bone interfaces which can compromise the mechanical properties of the tissue. One reason for this is the formation of fibrous adhesions [19]. Fibrous adhesions occur between the healing tendon and the surrounding tissues, and interfere with tendon gliding. This limits tendon excursion and reduces the functionality of the repaired tendon [41]. The organisation of the collagen fibre crimp